In various medical, chemical, biochemical or pharmaceutical applications the analysis of the spatial distribution and the dynamic redistribution of substances, i.e. molecules, atoms, molecular complexes and the like, in an assay sample, e.g. live or fixed cells, tissues and organs, is of importance. For example, temporal and spatial (re)distribution of molecules and molecular complexes is essential for biological processes in the human or animal body, such as the development of diseases or the action of drugs. In order to gather information about the (re)distribution of a substance in an assay sample, various “chemical imaging methods” are used that generate output images on a raster scale of millimeters, micrometers or nanometers.
For example, such chemical imaging methods are optical imaging of stained tissue sections or fluorescently labelled molecules in an assay sample, positron emission tomography, auto radiography, electron microscopy, and atomic force microscopy. While all of these methods are capable of generating multidimensional pictures with resolutions on the millimeter to nanometer scale, these methods produce rather limited chemical information which is often as important as the morphology of the analysed assay sample.
Further examples of chemical imaging methods producing rich chemical information are infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance based imaging. Although rich in chemical information, these methods usually lack sufficient sensitivity and spatial resolution for satisfyingly providing information about the temporal and spatial micro-distribution of substances in biological samples, particularly in cells, tissues and organs.
Another highly sensitive approach used for chemical imaging is imaging mass spectrometry. Various methods of imaging mass spectrometry have already been developed. Mass spectrometry is an analytical method that measures the mass-to-charge ratios of ions allowing the detection of known as well as of unknown substances. A general requirement for mass spectrometry analysis is that the substance to be analyzed has to be transferred into the gas phase and has to be ionized. This can for example be achieved by ion-beam-induced desorption, laser desorption or electrospray ionization. In imaging mass spectrometry, substances from a plurality of predefined spatial spots of an assay sample are transferred into the gas phase, ionized and then analyzed via mass spectrometry one after another. The results of the mass spectrometry analysis together with the spatial information of the spots can then be used to produce a chemical output image corresponding to the assay sample.
Some imaging mass spectrometry methods include for example ion-beam-induced desorption to perform ionization and sputtering of substances using a beam of high-energy ions. This ion beam is typically formed by means of an electric field and is impacted on the assay sample surface for inducing collisions. Thereby, some of the substances of the assay sample are ejected from the surface into the gas phase. Typically, ion-beam-induced desorption results in small fragment ions and atoms and is not suitable for imaging larger molecules, in particular biomolecules.
Other imaging mass spectrometry methods include laser desorption, where photons of a laser beam are used instead of the high-energy ions described above. Again, small fragment ions and atoms do result from laser desorption not enabling satisfying imaging of larger molecules. Particularly for imaging larger molecules, such as biomolecules, laser desorption has been further developed to matrix-assisted laser desorption ionization (MALDI). Therein, the assay sample is primarily coated by a matrix and certain substances are extracted into the matrix. Then an appropriate laser focus steps across the assay sample, typically in a raster pattern. The laser radiation is locally absorbed by the matrix leading the substances to be ionized and to be released from the matrix. Only little fragmentation of the substances occurs during this desorption process making matrix-assisted laser desorption ionization suitable for many chemical imaging applications. However, the development and selection of a matrix material suitable for desorption of a broad variety of substances is a difficult task. Additionally, the matrix is often not vertically extracting the substances out of the assay sample and horizontal diffusion of the molecules occurs inside the matrix. Finally, the volume of matrix plasma to be generated for ionization of biomolecules can not be made infinitely small, ion extraction starts only after a finite volume threshold. These effects deteriorate spatial resolution of chemical imaging methods using imaging mass spectrometry with matrix-assisted laser desorption ionization.
Still further, imaging mass spectrometry methods can also include the use of high voltage for extracting substances from the assay sample and for retaining the substances on an extractor. This use of high voltage results in fragmented molecules and is also not suitable for chemical imaging of larger molecules, in particular of biomolecules.
In addition to the described ionization methods that are used in imaging mass spectrometry, additional ionization methods are known that have not yet found application for imaging of biomaterials. These methods include electrospray ionization, where an aerosol of highly ionized droplets composed of volatile solvents and non-volatile analyte substances is formed in an electric field. The droplets are subsequently reduced in size by a combination of solvent evaporation and solvent coulombic explosions until ionized substances in gas phase result. Electrospray ionization causes only little substance fragmentation. However, for assay samples with high concentrations of inorganic salts, detergents or other non-volatile substances how they occur in tissue slices and other biomaterials mass spectrometry methods with direct electrospray ionization are not suitable.
Therefore there is a need for a device enabling an economic and exact chemical imaging of comparably large molecules, particularly biomolecules in assay samples.